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Biochimie (1996) 78, 518-529 Soci~t~franqaise de biochim ie et biologie mol6culaire Elsevier, Paris

Edit ing and import : S trateg ies for prov id ing p lant mitochondr iawith a complete se t o f funct iona l transfer RNAsA D i e t r i c h a , I S m a l l b , A C o s s e t a , J H W e l l a , L M a r 6 c h a l - D r o u a r d a

alnstitut de Biolbogie M ol6culaire des Plantes du CNRS, Universitd Louis-Pastelo; 12, rue du G~n~ral-Zimmo; F-67084 Strasbourg cedex;Station d e Gdn~tique et d'Amdlioration des Plantes. INRA, Route de St-CyJ; 78026 Versailles cedex, France(Received 24 April 1996; accepted 7 August 1996)

Su m m ary - - The recombinations and mutations that p lant mitochondr ial DNA has undergone dur ing evolution have led to the inactivationor complete loss o f a num ber of the 'nat ive ' t ransfer RNA genes der iv ing f rom the genom e o f the ancestral endosymbiont. Following sequencedivergence in their genes, some native mitochondr ial tRNAs are ' rescued ' by edit ing , a post-transcrip t ional p . rocess whic h changes the RNAl"Ne 111Spr imary sequence. According to in vitro s tudies with the native mitochondr ial tR N A from potato and tR N A from larch , ed it ing is required

f o r eff icient processing . Som e of the native tRN A genes which have been inactivated or lost have been replaced by tRN A genes present inplastid DNA sequences acquired by the mitochondrial genome during evolution, which raises the problem of the transcriptional regulationof tRNA genes in p lant mitochondr ia. Finally , tRNAs for which no gene is present in the mitochondr ial genome are im por ted f rom the cytosolThis process is highly specific for certain tRN As, and it has bee n suggested that the cognate am inoacyl-tRN As]tnthetases may be responsiblefor this specificity. Indeed, a mutation w hich blocks reco gnition of the cytosolic Arabidopsis thaliana tRNA"*"*by the corresponding alanyl-tRNA synthetase also prevents mitochondrial import of this tRNA in transgenic plants. Conversely, no significant mitochondrial co-importof the norm ally cytosol-specific tRNAasp was detected in transgenic plants expressing the yea st cytosolic aspartyl-tRNA synthetase fused toa mitochon driai targeting sequence, suggesting that, although necessary, recognition by a cogna te aminoacyl-tRN A synthetase might not besuff icient to al low tR NA impor t in to p lant mitochondr ia.a m i n o a c y l - t R N A s y n t h e t a s e / c h l o r o p l a s t / e d i t i n g / e v o l u t i o n / i m p o r t / m i t o c h o n d r i a / p l a n t / t r a n s f e r R N A

I n t r o d u c t i o nA l t h o u g h r e c e n t d a t a s u g g e s t t h a t m i t o c h o n d r i a h a v e am o n o p h y l e t i c e n d o s y m b i o t i c o r i g i n I l l , t h e m i t o c h o n d r i a lg e n o m e o f h i g h e r p l a n t s i s e x c e p t i o n a l l y l a r ge c o m p a r e d t ot h a t o f o t h e r e u k a r y o t e s [ 2] , I t e x h i b i t s s t r u c t u r a l i n s t a b i l i t ya n d c o n s is t s o f s u b g e n o m i c m o l e c u l e s u s u a l l y a b l e t o r e -c o m b i n e v ia r e p e a t e d s e q u e n c e s [ 3 , 4 ] . T h e l a r g e s i z e o fp l an t m i t o c h o n d r i a l g e n o m e s i s a p p a r e n t ly o n l y p a r t ly d u et o a g r e a t e r g e n e c o n t e n t a n d m a i n l y d u e t o i n t e r s p e r s e dn o n - c o d i n g s e q u e n c e s o f v a r i e d a n d m o s t l y u n k n o w no r i g i n , a s w e l l a s t o i n s e r t i o n o f l a r g e a m o u n t s o f f o r e i g nD N A o f p l a s t i d a n d n u c l e a r o r i g i n [ 5 , 6 1. A l t h o u g h m o s tm i t o c h o n d r i a l p o ly p e p t i d e s a r e n u c l e a r l y e n c o d e d a n d i m -p o r e d , s o m e r i b o s o m a l p r o t e i n s a n d k e y p o l y p e p t i d e s i n -v o l : e d i n t h e e n z y m a t i c c o m p l e x e s o f t h e r e s p i r a to r y c h a i na r e s y n t h e s i z e d b y t h e o r g a n e l l e t r a n s l a t i o n m a c h i n e r y .H e n c e , p l a n t m i t o c h o n d r i a r e q u i r e a f u l l s e t o f f u n c t i o n a l

Abbreviations: AlaRS, alanyl-tRNA synthetase; A spRS, aspar ty l-tRNA syn thetase; LysR S, lysyl-tRNA synthetase; R T-PCR, reversetranscription-polymerase chain reaction.Note: Larix x leptoeuropaea results from the crossing of Lari.:kaempferi and Larix decidua.

t r a n sf e r R N A s ( t R N A s ) . G e n e r a l l y , m i t o c h o n d r i a l g e n o m e sc o n t a i n b e t w e e n 2 2 a n d 2 9 t R N A g e n e s I 7 - 1 0 ] , s u f f i c i e n tt o d e c o d e a l l c o d o n s g i v e n r e l a x e d b a s e - p a i r i n g r u l e s ( U : Nw o b b l e ) . I n c o n t r a s t , m i t o c h o n d r i a l g e n o m e s o f v a s c u l a rh i g h e r p la n t s c o n t a i n o n l y 1 0 - ! 2 ' n a t i v e ' ( ie d e r i v i n g f r o mt h e g e n o m e o f t h e a n c e s t r a l e n d o s y m b i o t i c b a c t e r i u m )t R N A g e n e s [ 1 1 - 1 3 ] . T h i s i s m a n i f e s t l y i n s u f f i c i e n t t o p r o -v i d e f o r t h e n e e d s o f t h e m i t o c h o n d r i a l t r a n s l a t i o n s ~ ,s te m ,a n d t h u s p l a n t m i t o c h o n d r i a r e l y o n a l t e r n a t i v e s o u r c e s o ft R N A s . S o m e o f t h e m i s s i n g n a t i v e g e n e s h a v e b e e n r e -p l a c e d b y f u n c t i o n a l t R N A g e n e s c a r r i e d b y c h l o r o p l a s tD N A f r a g m e n t s i n s e r t e d i n t o t h e m i t o c h o n d r i a l g e n o m ed u r i n g t h e c o u r s e o f e v o l u t io n (eg [ 14 - -16 ]) . Ta ken to ge the r ,t h e n a t i v e t R N A s a n d t h e s e s o - c a l l e d ' c h l o r o p l a s t - l i k e 't R N A s s t il l d o n o t a c c o u n t f o r t h e 2 0 a m i n o a c i d s fo u n d i np r o t e i n s. T h u s , a n u m b e r o f h i g h e r p l a n t m i t o c h o n d r i a lt R N A s a r e n o l o n g e r e n c o d e d b y t h e o r g a n e l l a r g e n o m e , b u ta r e e n c o d e d i n t h e n u c l e u s a n d i m p o r t e d f r o m t h e c y t o s o l[ 1 7 , 1 8 1 . F i n a l l y , a s a f u r t h e r p e c u l i a r f e a t u r e , i t r e c e n t l ya p p e a r e d t h a t , t o b e f u n c t i o n a l , s o m e o f t h e h i g h e r p l a n tn a t iv e m i t o c h o n d r i a l t R N A s n e e d t o b e m o d i f i e d b y e d it i n g ,a p o s t - t r a n s c r i p t i o n a l p r o c e s s w h i c h i n t r o d u c e s c h a n g e si n t o t h e R N A s e q u e n c e [ 1 9 , 2 0 ] .

T h e s e p r o c e s s e s , w h i c h t o g e t h e r p r o v id e p l a n t m i t o c h o n -d r i a w i th a c o m p l e t e s e t o f n o n - r e d u n d a n t f u n c t i o n a l tR N A s


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( th e c h l o r o p la s t - l ik e a n d n u c l e u s - e n c o d e d s p e c i e s d o n o th a v e n a t iv e c o u n t e r p a r t s r e c o g n i z i n g t h e s a m e c o d o n ) , r a i s ei n t r i g u i n g q u e s t i o n s r e g a r d i n g e v o l u t i o n , a n d re l y o n s o f a rp o o r l y u n d e r s t o o d f u n d a m e n t a l c e l l u l a r m e c h a n i s m s : s p e -c i fi c R N A s e q u e n c e c o n v e r s i o n , t r a n s f e r o f g e n e t ic i n f o r-m a t i o n b e t w e e n d i f f e r en t c o m p a r t m e n t s , r e g u la t io n o fm i t o c h o n d r i a l g e n e e x p r e s s i o n , t r a n s p o r t o f n u c l e i c a c i d st h r o u g h t h e d o u b l e m i t o c h o n d r i a l e n v e l o p e . T h e p r e s e n t a r-t ic l e re v i e w s t h e p r o g r e s s m a d e b y o u r g r o u p s d u r i n g t h el a st f e w y e a r s i n a d d r e s s i n g t h e s e p r o b l e m s . I t a l s o i n c l u d e so u r m o s t r e c e n t e x p e r i m e n t s u s i n g t r a n s g e n ic p la n ~s to a n a -l y z e m i t o c h o n d r i a i t R N A i m p o r t . O u r s t u d i e s o n t h e th r e ep l a n t m i t o c h o n d r i a l t R N A c l a s s e s ( n a t i v e , c h lo r o p l a s t -l i k e ,n u c l e u s - e n c o d e d ) a i m a t a b e t t e r u n d e r s t a n d in g o f b o the v o l u t i o n a r y e v e n t s a n d f u n c t i o n a l p r o c e s s e s a n d m a yp o t e n t i a l l y h a v e p r a c t i c a l a p p l i c a t i o n s .

Materials and methodsIsolation o f ~ytoplasmic tRNAs and aminoacylatimt assaysF o r a mi n o a c y l a t i o n s t u d i e s, to t al c y t o p l a s m i c t RNA wa s e x t r a c t e df r o m p o t a t o t u b e r s b y p h e n o l e x t r a c t i o n a n d r e c o v e r e d b y e t h a n o lp r e c i p i ta t i o n 1 2 1 ] . La r g e RN As we r e e l i mi n a t e d i n t h e p r e s e n c e o fl M Na C! a n d t RNAs we r e f u r t h e r p u r i f i e d b y DEAE- c e l l u l o s ec h r o m a t o g r a p h y 1 2 2 1. Th e p r o p o r t i o n o f m i t o c h o n d r i a l ma t e r i a l i ns u c h p r e p a r a t i o n s u s u a l l y d o e s n o t e x c e e d 0 . 5 %.Am i n o a c y l a t i o n o f to t al p o t a t o c y t o p l a s m i c t RNA w a s c o n d u c t e da t 3 7 C i n t h e p r e s e n c e o f p u ri fi e d y e a s t a s p a r t y l - tRN A s y n t h e t a s e(Asp RS ) . Th e aminoacyla t ion reac t ion mixture conta ined 50 m MTris-HCl buffe r (pH 7 .5) , 10 mM ATP, 15 m M MgCI_,, 0 .4 m M glu-ta th ione , 0 .1 m g/m L bovin e serum a lbumin and 25 ~tM L-I 3H laspar-l ic ac id ( I so topchim , France) a t 6 .5 Ci /mm ole . Pure yeas t AspR S 1231wa s a g i l i f r o m J e a n Ga n g l o f f a n d G i l b e r t E r ia n i. E n z y me a c t i v it ycont ro l tes ts involved commercia l yeas t to tal IRNA .DNA co nstrm'tsTh e c o d i n g s e q u e n c e t o r t he y e a s t c y t o s o l i c As p RS I i 'o m t h e p l a s -mi d p TG 9 0 8 A 7 0 1 2 41 Ca g i l t f r o m J e a n G a n g lo f f~ wa s c l o n e d i n at ransla tional fus ion behind a sequen ce encod ing the f ir s t 90 am inoa c i d s o f t h e l ~ -s u b u ni t o f t h e mi t o c h o n d r i a l ATP s y n t h a s e f r o mNicotiana plumbaginifidia 125 I . This region inc ludes the mi toc hon -d r ia l t a r g et in g s e q u e n c e . Th e e x p r e s s i o n o f t h e f u si o n g e n e w a sc o n t r o l l e d b y t h e p r o m o t e r f r o m t h e c l a s s I p at a ti n g e n e B 3 3 1 2 6 1a n d t h e p o l y A s i g n a l f r o m c a u l i f l o we r mo s a i c v i r u s , t a k e n f r o mt h e p l a s mi d p A BD I 1 2 7 1 , Th e v e c t o r u s e d f o r th e s e e x p e r i me n t swas Bluescr ip t KS+ (St ra tagene) .Plant transformationS i x t y la g o f t h e p l a s mi d c o n t a i n i n g t h e A s p R S f u s i o n g e n e a n d 5 0l ag o f p A BD ! ( c on t ai n in g a n e o m y c i n p h o s p h o t r a n s f e r a s e g e n e[ 2 7 ] ) we r e u s e d t o t r a n s f o ml 2 x 1 0 " p o t a t o p r o t o p l a s t s ( p o t a t oc l o n e 2 i 7 , a c r o s s o f Solanum tuberosum a n d Solamtm chacoense).Protop las t prepara t ion , e lec t ropo ra t ion and regenera t ion of trans-g e n i c p l a n t s w a s a s d e s c r i b e d b y M a s s o n et al 1281. An t ib io t icr e s i s t a n t p l a n t s we r e s c r e e n e d f o r t r a n s c r i p t s f r o m t h e As p RS f u -

5 1 9s ion gene by R Na se prolec t ion to ident ify those p lants '~ hM~ hadintegra ted the A spR S fus ion gen e as wel l as the res is tance marker .Isolation d'mitochondria fi'mn transgenic planLsMflochondr ia f rom t ransgenic pota to tubers and leaves were i so-la ted on P ercol l gradients [291 and sub mit ted to an osmot ic shockt o d is r u p t t h e o u t e r me m b r a n e . F o r t h a t p u r p o s e , m i t oc h o n d ri a we r eresuspended in 50 la l i so tonic buf fer and subsequent ly d i lu ted in650 ~1 s te r i le water . Af ter 30 s , the osm ot ic pressu re was readjus tedus ing 650 ta l of two t imes concent ra ted buffer . To remove res idualp r o te in c o n t a mi n a t i o n , m i t o p l a s t s we r e i n c u b a t e d f o r 3 0 mi n o n i c ei n th e p r e s e n c e o f 0 . 2 5 mg / m L p r o t e i n a s e K a n d f i n al ly r e c o v e r e db y c e n t r i f u g a t i o n ( 3 mi n a t 8 0 0 0 g ) a f t e r a d d i t i o n o f i mM p h e -n y l me t h y l s u l f o n y l f l u o r i d e a s a p r o t e a s e i n h ib i to r . To r e m o v e r e s i d -ua l RNA contam inat ion , m i toplas ts w ere incubated for 5 min a t2 3 C i n t h e p r e s e n c e o f a n RNa s e mi x t u r e ( 4 0 p . g / mL RNa s e A ,4 0 0 u n i t s / mL RNa s e T I , I u n i t / mL p h o s p h o d i e s t e r a s e ) a n d r e -c o v e r e d b y c e n t r i f u g a t io n .Western blot analysisFor Western b lo t ana lys is , pro te inase K- t rea ted mi toplas ts werel y s e d in a [ 0 m M Tr i s - HCl ( p H 7 . 5 ), ! mM EDTA. 2 % ( v / v ) T r it o nX-10 0 buffer . Af ter addi tion of I volu m e of denaturing buffer 1301s a mp l e s we r e c e n t r i f u g e d f o r 1 5 mi n a t 5 0 0 0 0 g a n d t he s u p e m a -r a nt wa s l o a d e d o n 1 2 % ( w/ v ) d e n at u ri n g p o l y a c r y l a mi d e g e l s [ 3 0 ].P u r e y e a s t As p RS wa s a l s o l o a d e d o n t h e g e l s a s a c o n tr o l, a s we l las molecular mass prote in markers (Sigma) . Af ter migra t ion , ge lswe r e e l e c t ro t r a n s f e r r e d o n t o n y l o n m e mb r a n e s ( l mmo b i lo n - P , Mi l -l ipore) for 90 ra in a t 500 m A in 25 mM Tr is , 192 mM glycine , 15%( v / v ) me t h a n o l . No n - s p e c i f i c b i n d i n g wa s b l o c k e d b y i n c u b a t i o nIbr 2 h a t 37C in 10 mM Tr is -HCI (pH 7 .5) , 150 mM NaCI , 0 .05%( v / v ) Twe e n - 2 0 , 5 % ( w/ v ) d r y mi l k a n d 5 % ( v / v ) n o r ma l g o a tserum. Incubat ion wi th t i le polyc lonal rabbi t an t i serum agains ty e a s t As p RS 1 3 1 1 ( a g i ft f r o m J e a n G a n g l o f f ) w a s f o r 2 h a t r o o mtempera ture wi th a 1 /10 000 (v /v) f ina l d i lu t ion in the b lockingme d i u m . B i n d i n g o f t h e p r ima r y a n t i b o d y w a s re v e al ed b y c h e m i -ca l luminescence us ing a peroxidase-conjugated ant i serum agains lr a b b i t l g Gs a n d ECL r e a g e n t s ( Ame r s h a m) .I so la t ion o f m i tochondr ia l tRNAsTo t al m i t o c h o n d r i a l t RN A w a s e x t ra c t e d f r o m RN a s e - d i g e s t e d mi -toplas ts by phenol ext rac tion and recov ered by e thanol prec ip i ta -t i o n a s p r e v i o u s l y d e s c r i b e d [ 2 1 J . F o r N o r t h e r n b l o t t i n g ,mi t o c h o n d r i a l t RN As w e r e f ra c t i o n a t e d b y e l e c t r o p h o r e s is o n 1 5 %( w / v ) p o l y a c r y l a m i d e g e l s u n d e r d e n a t u r i n g c o n d i t i o n s [ 3 2 1 .Nor thern analy s is wa s as descr ib ed [33 I .

Results and discussionW e h a v e a d d r e s s e d d i f fe r e n t q u e s t i o n s r a i s e d b y th e c o m -p l e x g e n e t i c o r i g i n o f p l a n t m i t o c h o n d r i a l t R N A s , i n c l u d i n ge v o l u t i o n o f tR N A g e n e s i n t h e p la n t m i t o c h o n d r i a lg e n o l n e s , e d i t i n g a n ti t R N A f u n c t i o n , r e g u l a t i o n o f th e e x -p r e s s io n o f b o t h n a t iv e a n d c h l o r o p l a s t- l ik e t R N A g e n e s i np l a n t m i t o c h o n d r i a , a s w e ll a s t R N A t a rg e t in g a n d i m p o r ti n to p l a n t m i t o c h o n d r i a . A s a l r e a d y o b v i o u s f r o m p r e v i o u s


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520reports (e g [11,18]) and fur the r shown by our recent work[34], important differences in the chloroplast-l ike and im-ported tRNA spec ies appear be tween and wi th in p lant l i -neages. Thus , tRN A gene evolut ion jus t i f ie s the choice ofthe piant/tRNA system s studied and is present as a backd ropto mo re func t iona l aspec ts throughout the p resent work. Themain sec t ions a re organized a round the three c lasses of p lantmi tochondria l tRNA s, na t ive , chloroplas t- l ike and nu c leus-e nc ode d , wi t h e mpha s i s on e d i t i ng , c on t ro l o f ge ne e x -pression and import mechanisms, respectively.Edit ing as a strategy to yield f imction al nativemitochondrial tRNAsA firs t s t ra tegy deve loped by p lant m i tochondria to keep acomplete tRNA set is to correct a t the RNA level l imitedsequence d ivergence wh ich has occurred in some of the na-t ive m i tochondria l tRN A genes . This i s achieved by edi t ing ,a w idespread post-transcriptional proc ess first identified inkinetoplast ids from Trypanosoma brucei [35] and whichchanges the pr imary sequence of RNA s, as com pared to tha tof the corresponding DNA templa tes ( for reviews see eg[36--38]). It is mainly ob served in m itocho ndria , to a lesserextent in chloroplasts and in a few cases in transcripts ofnuc lea r genes . In h igher p lant mi tocho ndria and in chloro-plasts , RNA edit ing results general ly in the conversion ofcytidines to uridines. Although edit ing was first found al-most exc lus ive ly in messenger RNA s, w here i t cont r ibutesto the conservation of functional proteins, edit ing eventsaffect ing ribo som al RN A, intron sequen ces and tRNAs hav enow been observed in va r ious organism, , , ( re fe rences in[201). As des cribed below, i t appears in part icular that som ehigher plant nat ive mitochond rial tRN As w ould not be func-t ional in the absen ce of editing.Potato and b ean mitochondrial native tRN A m,,"and larchmimchondrial native tRNAHisBy comparing the sequences of the bean (Phaseolus vul.garis) and pota to (Solanum tuberosum) native mitochon-drial tRNA Ph~ gene s to those o f the corresp ond ing m aturetRNAs, we dem onst ra ted tha t in both d icotyledo nous an-giosperms (flow ering plants) the C enc od ed at posi t ion 4 inthe gene i s conver ted in to a U in the mature tRN A [391. Thisnuc leot ide change correc ts the m ismatched C4:A69 base-pa i r which appears wh en fo ld ing the gene sequ ence in to thec lover lea f s truc ture . A minoacyla t ion of in vitro synthes izedune d i t e d a nd e d i t e d po t a t o t RNAP he t r a nsc r i p t s i n t hepresence of a p lant mi tochondria l enzymat ic ext rac t show edthat edit ing h as no significant influe nce o n tRNA Pae charg-ing with phenylalanine. However, RT-PCR amplificat ionsof na tura l p ota to mi tochon dria l tRNAP~,e precursors andproc e s s i ng a s sa ys wi t h in v i t ro synthes ized precursorsshowed that Ca to U4 edit ing is a prerequisi te for effic ientprocessing o f potato nat ive mitoc hon drial tRNAPhe [20]. In-deed, no unedi ted tRNAPhe has been found in vivo at thede tec t ion leve l of pur i f ica t ion and di rec t sequenc ing [39].

Based on sequence data , we speculated [20] that a U4:A6,W a t son -Cr i c k ba se -pa i r i ng i s e s se n t i a l t o ma i n t a i n t hec lover lea f fo ld ing of the tRNA part in the pota to mi tochon-dr ia l tRNA Phe precu rsor RNA and to avoid an a l te rna tes tem-loop fo ld ing involving the ups t ream region of the pre -cursor which i s unl ike ly to be recognized by RN Ase E C4to U4 edi t ing a l so occurs in the mi tochondria l tRNA P~e o foenothera (Oenothera berteriana), another d icotyledonousangiosperm, and i s a l so requi red for process ing [19, 40].Similarly, folding the sequence of the nat ive mitochon-drial tRNA His gen e expreszed in larch (Larix x leptoeuro-pae a , a g y m n o s p e r m ; g y m n o s p e r m s c o n s t i t u t e a l e s sevolved group of v ascula r plants than angiosperm s and a remainly represented by con i fe rs ) in to the c lov er lea f s t ruc tureyields three C :A mism atched b ase-pairs (C6:A67, CI2:A23and A29:C41using c lass ica l tRNA nuc lee f ide numbering)Again sequenc ing of tRNA ni~ precursor molecules and invitro process ing assays dem onst ra ted tha t al l three C:A mis-pairings are edited into classical U:A base-pairs and thatuned ited tRNAHi~ precurso rs are not p rocess ed [41].Native m itocho ndria l tRNA Hi~ and tRN A Ph~ are found inl ive rwort (Matz 'hantia polymorpha, a bryo phyte ) [8] . In th i snon-vascula r pr imi t ive p lant , no edi t ing has ever been de-tected and a U, instead of a C, is fou nd at the gen e level inal l the posi t ions mentioned above as edit ing si tes in thelarch tRNAHi.~ and potato tRN A Phe. Edit in g therefore ap-pears to correc t the sequ ence of some n a t ive p lant mi tochon-dr ia l tRNAs fol lowing sequence dr i f t in the i r genes andthereby ' re scues ' these tRNAs which would o therwise benon-func t iona l .Potato mitochondrial nqtive tRNAn,'Original ly , the 1erm 'RNA edi t ing ' re fe rred to pr imary se -quence changes in messeng er RNAs leading to codon modi -ficat ion 1351. To defin e the l im its of w hat sh ould be c al lededi t ing when tKN As a re concerned i s m uch less obvious , asthey un dergo a ,~a r ie ty of posH ranscr ip t iona l m odi f ica tionsin addi tion to pr imary sequence changes . The C to U t ran-s it ions in p lant m i tochondria l RNAs a re l ike ly to occur vi aa s imple deam ina t ion reac t ion [42, 431, a process w hich i scomparable to many other nuc leot ide modi f ica t ion reac-t ions occurr ing in tRNAs. A lso , many n uc leot ide modi f ica -t ions in tRNAs can be considered to be as si te-specific asthe pr imary sequence changes d iscussed above . I t wouldthere fore be conce ivab le to ca l l ' tRN A edi t ing ' a l l nuc leo-t ide modi f ica t ions wi th c lea r func t iona l consequences . Onepos t - t ranscr ip t iona l modi f ica t ion of the pota to mi tochon-drial nat ive tRNAne would obviously fal l into such an ex-t e n d e d v i e w o f e d i t i n g . T h e m i t o c h o n d r i a l g e n ecorresponding to th i s tRNA conta ins a C res idue a t the f irs tpos it ion of the ant icodon and codes the re fore for a meth-ionine-specific (CAU) anticodon. The C is post-transcrip-t i ona l l y mod i f i e d so t ha t t he ma t u re t RNA c on t a i ns aiys id ine- like nuc leot ide (L*, a C res idue m odi f ied wi th anunident i f ied der iva t ive of the amino ac id lys ine) a t tha t po-si t ion and caL, be am inoa cylated w ith isoleu cine but not w ith


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m e t h i o n i n e [ 4 4 ] . A s f o r E. co t i t R N A ne ( L A U ) [ 4 5] t h e a m i -n o a c y l a t i o n a n d c o d o n s p e c i f i c i t y o f t h e p o t a t o m i t o c h o n -d r i a l n a t i v e t R N A n e ( L * A U ) i s li k e l y t o r e l y o n t h e ~ e d i t i n g 'o f C 3 4 in t o L ' 3 4 . T h i s p r o c e s s m i g h t o c c u r i n a ll ' p r o k a r y o -t i c ' s y s t e m s , a s a s i m i t a r t R N A ~le i s n o t o n l y p r e s e n t i n b a c -t er ia a n d m i t o c h o n d r i a o f d i c o t y l e d o n o u s a n g i o s p e r m s , b u tp r o b a b l y a l so i n m i t o c h o n d r i a o f m o n o c o t y l e d o n o u s a n -g i o s p e r m s l i k e w h e a t ( T r i t i c u m a e s t i v u m ) [ 1 5 1 a n d m a i z e( Z e a m a y s ) [ 4 6 ], i n m i t o c h o n d r i a o f t h e g y m n o s p e r m l a rc h[ 3 4 ] a n d i n c h l o r o p l a s t s [ 4 7 ] .R e p l a c e m e n t o f n a t i v e m i to c h o n d r i a l t R N A g e n e s b yc h l o r o p l a s t- d e r i v e d t R N A g e n e sE x p r e s s i o n o f c h l o r o p l a s t - o r i g i n a t i n g g e n e s p r e s e n t o np r o m i s c u o u s p l a s t id D N A f r a g m e n t s i n s e r te d i n t o t h e m i t o -c h o n d r i a l g e n o m e i n t h e c o u r s e o f e v o l u t i o n i s a s e c o n ds t r a t e g y u s e d b y p l a n t m i t o c h o n d r i a t o c o m p l e m e n t t h e i rt R N A s e t. T h e i s o l a ti o n f r o m b e a n m i t o c h o n d r i a o f a m ~ . tu r ec h l o r o p l a s t - l i k e t R N A ' r r p c l e a r l y d i s t i n g u i s h a b l e f r o v ~ i t sb e a n c h l o r o p l a s t c o u n t e r p a r t [ 1 4 ] w a s t h e f i r s t d i r e c t e v i -d e n c e f o r t h e e x p r e s s i o n o f a c h l o r o p l a s t - l i k e t R N A g e n e i nm i t o c h o n d r i a . C h l o r o p l a s t- l ik e t R N A g e n e s h av e b e e nf o u n d i n a n u m b e r o f d i f f e r en t p l a n t s [ 4 8 ] , s h o w i n g th ole h i sp h e n o m e n o n is g e n e ra l ( t a bl e I ). A l t h o u g h s o m e o f t h e s eg e n e s m a y h a v e i n a c t i v a ti n g m u t a t i o n s , m o s t h a v e r e m a i n e di d e n t ic a l o r a l m o s t i d e n t ic a l t o t h e i r a u t h e n t i c c h l o r o p l a s tc o u n t e r p a r t s . O n e o f t h e m a i n p r o b l e m s r a i s ed b y t h i s s i tu -a t i o n i s to u n d e r s t a n d h o w t h e t r a n s c r i p t i o n o f t h e s e p l a st i dg e n e s i s c o n t r o l l e d i n m i t o c h o n d r i a .Chloro plast - l ike tRN A eh,' , tRNACy~~a n d t R N A m ~I n c o n t r a s t t o d i c o t y l e d o n o u r ; p l a n t s l i k e p o t a t o , b e a n o ro e n o t h e r a , t h e n a t i v e t R N A eh~ g e n e i s a p s e u d o g e n e i n m i -t o c h o n d r i a o f t h e m o n o c o t y l e d o n o u s p la n t s w h e a t [ 4 9 ] a n dm a i z e ! 16 1 ( ta b l e I ) . W h e n t h e s e q u e n c e s o f t h e s e p s e u -d o g e n e s a r e l b l d e d i n t o t h e c l o v e r l e a f s t r u c t u r e , a C 4 - G t , 9b a s e - p a i r i s f o u n d i n s te a d o f t h e C4-A69 m i s p a i ri n g d e d u c e df r o m t h e b e a n , p o t a t o a n d o e n o t h e r a g e n e s e q u e n c e s . P r o -c e s s i n g o f p o t a t o p r e c u r s o r t R N A Phe c o n t a i n i n g a C4-G69b a s e - p a i r , i n s t e a d o f t h e U 4 - A 6 9 b a s e - p a i r n o r m a l l y p r o -d u c e d b y e d i t i n g , i s o n l y 4 0 % e f f i c i e n t I 2 0 ]. I t c an t h e r e f o r eb e s p e c u l a t e d t h a t a n A 6 9 t o G69 m u t a t i o n a p p e a r e d i n t h en a t iv e tR N A Ph e g e n e o f m o n o c o t y l e d o n o u s a n g i o s p e r m sa f t e r t h e i r d i v e r g e n c e f r o m d i c o t y l e d o n o u s a n g i o s p e r m s ,g i v i n g r i s e t o a t R N A Phe t r a n s c r i p t w h i c h c o u l d n o l o n g e rb e e d i t e d a n d w a s i n e f f i c i e n t ly p r o c e s s e d . I n a f u r t h e r e v o l -u t i o n a r y s t e p , t h e t r a n s fe r o f t h e c h l o r o p l a s t t R N A ph~ g e n ei n to t h e m i t o c h o n d r i a l g e n o m e o f m o n o c o t y l e d o n o u s p la n t s[ 1 5] m i g h t h a v e e n a b l e d e f f i c i e n t e x p r e s s i o n a n d p r o c e s s i n go f a n a l t e r n a t i v e t R N A P h ~ , l e a d i n g t o f u r t h e r d i v e r g e n c e o ft h e n a t i v e t R N A eh~ g e n e i n t o a p s e u d o g e n e . A s i m i l a r s e -q u e n c e o f e v e n t s m i g h t h a v e h a p p e n e d t o t h e m i t o c h o n d r i a ln a t i v e t R N A C y ~ w h i c h i s s ti l l e x p r e s s e d a n d , a t l e a s t i n o e n o -t h e r a , e d i t e d i n d i c o t y l e d o n o u s p l a n t s I ! 8 , 4 0 ] , b u t h a s b e e nr e p l a c e d b y t h e p l a s t id - d e r i v e d t R N A C y ~ i n t h e m i t o c h o n d r i a

521Tab le I , The origin o f tRNAs in different plan t groups . N, native;C, chloroplast-like; I, imported; ?, unknown. * indicates IRNAswhich are C-to-U edited. The data in this table are drawn mosllyfrom [8, 85] (liverwort), [! 3, 151 (mon ocotyledonous plants), [11,12, t8] (dicotyled onou s plants ) and [341 ( larch, monocotyle-dono us plants , dicotyledo nous plants ) .IR NA L i ve pw or t La rc h Di co ty l edonous M onoco ty ledonouplants s plantsAla N I I IArg N '~ 1 !A sn N 9 C CA sp N N N NCys N '~ N* CG i n N N N NG l u N N N NGiy N 1 N IHis N N* C C (I~al i e N / I b N / I b N / I b N / ! ~Leu N I I ILys N I N NMet N 9 N/C c N/CcPhe N I N* CPro N I N NSer N l N/Cd (l) e N (C)dThr N/I f 1 I IT r p N C C CT y r N N N NVal N I I (N?) g IaWheat m itochon dria import tRNA His [34]. btRNAtle (L*AU) isa native tR NA in all plants [48] , tRNA 1re (IAU) is imported [1 8,34, 86] . CtRNAnVlet s native and tRNA manet s chlorop)ast-like inm onoco tyledon ous and dicotyledonous plants [48]. UM itochon-dria of wheat and dicotyledonous plants, except sunflow er, expressa chloroplast-like RN Aaer (GG A) 1481 hat is absent from m aize [ 131.eSunflower mitochondria import tRN As~er, unlike .mitoch ond riao fother dicotyledonous ~lants exam ined to date 1341. At least one nu-cleus-encoded tRNA TM is imported into liverwort mitochondria(A ka sh i K, ~,laa~camaK, p ersonal comm unication), gPotatomitochon-drial tRNA s ' hybridize to mitochon drial DN A [18].

o f m o n o c o t y l e d o n o u s p l a n ts [ 1 5, 1 6] . M o r e o v e r , a c h l o r o -p l a s t - l i k e t R N A C y s p s e u d o g e n e s e e m s t o b e p r e s e n t i n t h ep o t a t o m i t o c h o n d r i a l g e n o m e [ 1 8 ] . F i n a l l y , t h e n a t i v et R N A tti~ p r e s e n t i n t h e b r y o p h y t e l i v e r w o r t [ 8 ] , a n d e x -p r e s s e d a n d e d i t e d i n t h e g y m n o s p e r m l a rc h [ 4 1 ] i s a b s e n tf r o m a n g i o s p e r m s w h e r e i t h a s b e e n r e p l a c e d i n m o s t c a s e sb y a c h l o r o p l a s t - l i k e t R N A r iis [ 1 8 , 5 0 , 5 1 ] . A g a i n , l i v e r w o r ta p p e a r s t o b e t h e s i m p l e s t s y s t e m s t u d i e d s o f a r , a s i t s m i -t o c h o n d r i a c o n t a i n n o p l a s t i d - d e r iv e d t R N A s [ 8] . I t m u s t b es t r e s s e d , h o w e v e r , t h a t f o r o t h e r p l a n t s p e c i e s t h e s i t u a t i o nh a s e v o l v e d d i f f e r e n t l y , a s n u c l e a r l y - e n c o d e d t R N A TM isu s e d i n l a r c h m i t o c h o n d r i a [ 3 4 ] , a n d t R N A H is i s i m p o r t e df r o m t h e c y t o s o l i n t o w h e a t m i t o c h o n d r i a [ 4 1 ] .


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52 2Transcriptional regulation of m itochondrial tRNA genesThe exis tence of both nat ive and chloroplas t- l ike tRNAgenes in p lant mitochondr ia makes their t ranscr ip t ionalregulation interesting. Moreover, whereas the coding se-quences of p las t id-derived tRNA genes have of ten remainedalmost unchanged s ince their in tegrat ion in to the m itochon-drial DNA, variou s rearrangements and/or se quen ce alter-ations have frequently occurred in their f lanking regions(fig 1). No t all of these plastid-derived tRNA gene s are ex -pressed. Alth oug h attempts ha ve been m ade [52], no abso-lu te ru le can be p roposed to p red ic t f rom the genera lsequence features w hether a chloroplas t- l ike tRNA genepresent in mitochon dr ia will be expressed or not . We haveisolated two id entical potato mitoc hond rial chloropla st-liketRNAAs. genes which exhibi t very dif ferent f lanking re-gions in their near vicinity, highly similar to the correspo nd-ing authentic chlo roplast regions in on e case and unrela tedto ch lo rop las t s equences in the o ther case (Mar6cha l -Drouard L, D esprez T, Chavant L, Dietrich A, in prepara-t ion; access ion numbers in the EMB L nucleot ide sequenced a t a b a s e X 9 3 5 7 5 a n d X 9 3 5 7 6 ) . T h e p r e s e n c e o f t h etRNAAshgene cop y s t il l f lanked by sequences of chloroplas ttype seem s to be restricted to som e Solanaceae. Similarly,although the chloroplast-like m itocho ndrial tRN A Hi~ gen eis identical or alm ost identical in pota to (Mar~chal-DrouardL, Desprez I' , Chavant L, Dietrich A, in preparation; ac-cess ion number in the EMBL nucleot ide sequence databaseX93577), oenothera [53], sunflower (Helianthus annuus,another dicotyle don ous plant) [52] and m aize [501, and th eregions flanking th is gene are h ighly s imilar between po-tato, oenothera and sunflower, the chloroplast sequencesm ain ta ined in the v ic in i ty o f the m aize m i tochondr ia ltRNA His gen e are com pletely unrelated. As all these ch loro-plast-like tRNA a,, . and tRNAHi~ gen es are active, it se em sthat their expression in plant mitoc hon dria is not depend entupon their imm ediate flanking sequ ences.In chloroplasts , expression of tRNA genes relies upon'proka ryot ic- l ik e ' -10 and -35 boxes , in ternal promoters , orco-transcription with other gen es (for a review see eg [54]).Knowledge about promoter sequences of tRNA genes inplant mitoch ondria is quite sparse at the present s tage. Thetranscription initiation site of the oeno thera mitochon drialnative tRNAOhe (GA A) g ene, w hich is probably co-tran-scribed w ith tRNA ho (UGG ), has be en the be st s tudied sofar. Th is transcription initiation site , locate d in an upstr eamp u r i n e r i c h r e g i o n c o n t a i n i n g t h e c o n s e n s u s m o t i fCRTAaGaGA found in putat ive mitochondr ial messen gerRNA and r ibosom al RNA prom oter s o f d ico ty ledonousplants [55, 56], was shown to be active in a pea (Pisumsativum) mitochondr ial in vi tro transcription system [ 5 7 1 .How ever , in po ta to m i toc hond r ia , the na t ive tRNA s~(GCU), tRNAPhe (GA A) and tRNA Pro (UG G) are c o-tran-scribed and the above men tioned consensus motif is notpresent at the initiation site of this transcription unit [581.Thus, no clear picture emerges yet and it remains to bedef ined to what extent chloroplast- l ike tRNA genes are ex-

pressed in plant m itocho ndria thank s to appropriate pro -mo ter sequ ence s or to inse rtion into already transcribed re-gions of the p lant m itochondr ial genom es . In th is contextit is interesting to note that, in all angiospe rm mitoc hond riastudied so far, the chlo roplast-like tRNATq gene is alwaysexpressed; in contras t , a chloroplas t- l ike tRNA Pro gen e,usually loc ated about 150 nt upstream of this tRNArrp gen ehas never been or is no long er expressed [59, 60].Replacement of native mitochondrial tRNAs by import ofnuclearly encoded species from the cytosolThe th ird s trategy used by plant mitochond r ia to comple-m en t the i r tRNA se t i s to share som e nuc leus -encodedspecies with the cy tosolic translation machinery. B eside theinabi li ty to f ind in p lant m itochondr ial genomes the mini-mal set of tRNA genes neede d to suppor t protein synthes is(even taking in to account the chloroplas t- like genes) (e g[11-13]) , evidence for tRNA imp ort f rom the cytosol cam efrom the presence in p lant m itochond r ia of ' cytosol- l iketRNAs hyb ridizin g to the nuclear gen om e [15, 17, 18, 32,61, 62]. I t appeared from sequence analysis that some nu-clearly enc ode d tRN As partition bet we en the cytosol andthe m itochondr ia , as the only dif ference ident i f ied betwee nthe cytosol ic and mitochondr ial forms o f a g iven tRNA wasthe post-transcriptional mo dification of G i8 into Gm ~s intRNAsTM [17, 32, 61, 62]. Most of our efforts aim at un der-standing the sele ction and transport m echa nism s involv edin this still intriguing process.Nuclearly encoded mitochondrial tRNA sDirect proof for mitochondrial import of a nuclearly en-coded tRNA was obtained in t ransgenic p lants . The b eannuclear gene encod ing tRNALeu (C*A A) [32] was in tro-duced into potato. Specific probin g show ed that the corre-sponding heterologous tRNA was expressed and was indeedpar t i t ioning between the cytosol ic and the mitochondr iafractions [331. By m utating the transgen e, a four base-pairinser tion could even be in troduced in to the ant icodon of thetRNA L~" with out c om prom ising mitoch ond rial import [33]Com par ison of the mitochondr ial tRNA populat ions oflarch (gym nosperm), potato and sun f lower (d icotyledonouangiosperms) , as wel l as maize and wheat (monocotyled o n o u s a n g i o s p e r m s ) s h o w e d a n u m b e r o f d i f f e r e n c e swithin or between plant l ineages in the ident i ty of the im-por ted species , sometimes between relat ively closely re-lated plan ts [34]. S om e tRNAs, n am ely tRNAAJa, RNAsLeutRNATM , turned out to be impor ted f rom the cytosol in to themitoc hon dria in all plants tested (table I) . Conversely, othertRNAs sys tematical ly appeared to be enco ded by the mito-chondr ia l DNA : tRNAAsp , tRN A o ln , tR NA ~ lu , tRNA ne(L*AU) and tRNATyr. At leas t some of these have pecu-liarities dating from their prokaryotic origins which mightmake them dif f icul t to replace b y typical ly eukaryotic nu-clearly -enc oded equivalents: tRN AGIn becau se glutam inyltRNACla der ives f rom g lu tam yl - tRN Ao'n (e g [63 ] ) and


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5 2 3

A trnP trnW

not expressed expressed

c p h ig h e r # a n tm t m a i z er a t w h e a tm t s u g a r b e e t

trnH~ . ~ . ~ . ~ . ~ . ~ . Z . ~ . ~ e . ~ c p h i g h e r p M n t

i ~ ~ ~ .... ~:, - ' ' l m ~ m m m l l m ~ i . ~ i i i . i . ~ i i . . . i l ra t s u n f lo w erp o t a t o

C trnN~ . " / ~ Z l ~ ~ , / ' 2 " 2 " 2 ' # ' # ' # ' 2 Z cp m g h e r pJa . t

trnY~ ~ i i i . . . . i m m ~ ~ l l ~ ~ ........................... W B ~ I I B I B I rnt ~ un flo wer,Oenothera, p o t a t o 1trnDi , . - ...... - ~ ~ i ~ rn t w h e a t~ Z Z 2 , ~ c Z ~ ra t p o ta t o 2

Fig 1. Evolutionary aspects of chloroplast-like tRNA genes inserted into plant mitochondrial gen omes. A. Organization o f the chloroplast-likePro TrptRNA ( t r nP ) and tRNA ( t r nW ) gene clusters present in the m itochondrial genome of m aize [87], wheat [60] and sugar beet ( B e t a v u l gar i s )[591, a.~ comp ared to the corresponding region o f the chloroplast g enom es. B. Organization of the region containing the chloroplast-liketRNAms gene ( t r nH ) in the mitochondriai ge,aome c,f maize I50], sunflower [52 ] and potato (Mar6chal-Drouard L, Desprez T, Chavant L,Dietrich A , in preparation; accession numb er in the EM BL nucleotide sequence database X93577), as compared to the corresponding regionof the chloroplast genom es. C. Organization o f the region(s) containing the chloroplast-like tRNAAsh gene(s ) ( t r nN ) in the mitochondrialgenome of wheat 188[, sunflower [89 ] and polato (M~ r~chal-Drouard L. Desprez T, Chavant U Dietrich A, in preparation; accession numbersin the EM BL nucleotide sequence database X935 75 and X935 76), as compared to the corresponding region of the ~lorop last genom es, hithe case o f potato m itochondria, two chloroplast-like t r nN genes, nu mbered 1 and 2, h ave been identified. The tRNA lyr gene ( t raY) , locateddownstream o f t r n N in sunflower, oenothera and potato ( t r n N n correspond s to a native tRNA sp ecies [90]. cp refers to the chloroplast genom e,mt to the mitochondrial genome. Black bo xes represent tRNA genes, hatched boxes correspond to chloroplast-like intergenic sequences anddotted boxes to native intergenic sequences. Arrows indicate the direction of transcription.


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52 4tRNA ne (L*A U) bec ause of its pecu liar post-transcriptional~nodification (see above). T he ob servations m ade sug gestthat the ability to im port each tRNA has b een acquired in-dependen t ly a t d i f f e ren t t im es du r ing the evo lu t ion o fhigher plants [34].General considerations on the plan t mitochondrial tRNAimport proces sWe. have isolated a n umber o f bean nuclear tRNA genesczdlng for species presumably impor ted in to mitochon dr ia(tRNATM a n d t R N ATM) or p resen t on ly in the cy toso l(tRNA r~o and tRNASer). Align me nt of th ese tRNA gene se-quences , of previous ly obtained tRNA sequences , as wel l asof sequ ences available in the literature, did n ot reveal anysequence or secondary structure motif which is conservedand character is t ic for the nuclear ly encoded tRNAs im-porte~ into plant mitochondria, eith er in the tRNAs them -selves, or in the ge ne flanking sequen ces. Therefore, thereis no indicatio n that there exist particular characteristics inthese tRNA s, or their precursors, w hich w ould be specificfor mitochondr ial ly impor ted tRNAs (Ramamonjisoa D,Kauffmann S , Choisne N, Green G, Small I , Marfchal-Drouard L, Dietrich A, in preparation; accession numbe rsin the E MBL nuc leo t ide s equence da tabase X98179 toX98184).This lack o f conserve d features, toge ther with the variedpattern o f tRNA s im ported in different plants (eg [341), sug-gests that each impo rted tRNA is recogniz ed by a specificfactor. It is difficu lt to b elieve that the re is a specific im portchann el for each tRNA. A mo re plausible hypothesis is theinvolvem ent of specific protein carriers with m itochondriaitargeting seque nces that would direct the relevant cytosolictRNAs to the protein import channel. The protein importapparatus has bee n implicated in tRN A im port in yeast 1641,and DN A oligo nuc leotides cross-linked to pep tide carrierscan be im por ted in to yeast and m amm alian mitochondr ia[65, 66], The search for natural carriers has centered onproteins capable o f tightly binding to and shie lding tRNAs.Mi tochondr ia l am inoacy l - tRNA syn the tases were sug-gested as ideal candidates for such a role at a very earlystage [67].Recognit ion by a cognate aminoacy l - tRN A syn the taseseems necessary b u t no t su ff ic ien t for tRNA impor t in tomitochondria in transgenic plan tsThe inv olvem ent of am inoacyl- tRNA synthetases in thetRNA import process has been tested, with contradictoryresults, for trypanosomatid and yeast mitochondria. Importof tRNAs into isolated Le i s h m a n ia mitochondr ia does notrequire adde d protein factors [68, 69], and n on-am inoacy-latable tRNAs or precursor tRNAs can be mitochondriallyimported in Le i s h m a n ia an d Tr v p a n o s o m a b r u c e i [70, 711.In contrast, im port o f tRNAt-r , (CU U) into yeast m itochon -dr ia only occurs fo l lowing aminoacylat ion by the cytosol iclysyl-tRNA synthetase (LysRS) and in the presence of the

precursor to the mitochondrial LysRS, which is a distincenzyme [72] . In Tr y p a n o s o m a b r u c e i , even heterologoutRNAs can be impor ted [73] . This sugges ts loose recognit ion rules of individual tRNAs by the impor t m achinery intrypanosomatids, althou gh binding to receptors in the mitochondr ial mem brane thanks to a def ined sequence in the Dloop has been prop osed [69] . In yeas t , select ion for imporis very strict, as only a single tRNA is imported [74]. Inplants , mitoc hon drial tRNA im port is also selective (e g [1718, 34]), imply ing specific recognitio n of the tRNAs to beimpo rted by o ne or several factors of the transport pathwayTo tes t whether aminoacyl- tRNA synthetases are involvedin the recogni t ion of tRNAs im por ted in to p lant mitochondr ia , we t ransformed plants with a heterologous gene en-coding a tRNA which is no longer recognize d by i ts cognateaminoacyl-tRNA synthetase and look ed at the fate of thecorrespo nding transcript.Expression an d mitochondrial import o f tRNA ata variantsin transgenic plan tsIn a l l h igher p lan t s s tud ied so f a r , the m i tochondr ia ltRNA Ala is a m;~Oearly enc ode d spe cies impo rted from thecytosol. In bacteria [75] and anim als [76], the major identitymo tif in tRNA Ala reco gniz ed by alany l-tRN A synthetase(AlaRS) is the G 3:U70 wo bble pair in the acceptor s tem.Res tor ing normal Watson-Crick base-pair ing by al ter ingUT0 o C70 abolish es bindin g of AIaRS to tR NA Ala and he nc eabol ishes aminoacylat ion. We have shown that the samemutat ion has an ident ical effect on the cytosol ic/mitochondrial tRNA Ala from plants, both by in vitro aminoacylat ionassays, and by am ber suppressor tests in v ivo [77]. The w ild-type (UT0) and the mutated (C70) Arabidops is tha l iana (ad ico ty ledonous ang iosperm ) tRN Aala were expres sed intransgenic tobacco (another d icotyledonous angiosperm)plants . W hereas both form s were present at a norm al levelin the cytosolic fraction, only the wild-type, aminoacylat-able tRNA ARa was recove red in the mitoc hond rial fraction[78]. A four-nucleotide insertion (TCGA) was also intro-duced in to the ant icodon loop of these U70/C70 tRNAA~constructs , with no s ignificant consequ ence on the aminoa-cylat ion proper t ies of the corresponding tRNAs. A gain , re-placement of UT0 with C70 led to the lack of the heterologoustRNAA~a n the mitoc hond rial fraction, wh ereas the insertionin the ant icodon loop had no apparent ef fect on the part i-t ioning between the two f ractions [78] . Com plementary invitro exper imen ts involving wild type or mutated tRNA Alatranscripts a nd toba cco mito cho ndria l extracts suggestedthat the absence of the G3:C70 RNAs in m itochondr ia of thetransgenic plants is unlikely to be due to differential s ta-bility versus the G3:UT0 tRNAs I 7 8 1 .It appears from these expe riments that a s ingle inactivat-ing base change is sufficient to abolish both amino acylationand mitoc hond rial import o f tRNAAI', . This implies that in-teraction with AIaRS is a prerequ isite for tRN A Ala impo rtin to p lant m itochondr ia . The plant tRNA Ala mp ort mecha n-ism is therefore likely to re sem ble that o f tRNALy~ (CUU)


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i n y e a s t [ 7 2 ] , a p a r t f r o m t h e f a c t t h a t t h e p l a n t c y t o s o l i c a n dm i t o c h o n d r i a l A I a R S a r e e n c o d e d b y t h e s a m e g e n e [ 7 9 ].F u r t h e r a t t e m p t s w e r e c a r r i e d o u t i n w h i c h t h e G 3 : U ? 0i d e n t i t y m o t i f o f tR N A A ~ a , a n i m p o r t e d t R N A , w a s i n t r o -d u c e d i n t o c y t o s o l i c I R N A P he , a t R N A w h i c h i s n o t i m p o r t e di n t o t h e m i t o c h o n d r i a o f th e a n g i o s p e r m s s t u d i e d s o f a r. T h ep h e n y l a l a n i n e t o a l a n i n e a m i n o a c y l a t i o n i d e n t i t y s w i t c h

w o r k e d r e a s o n a b l y w e l l w i t h t h e A th a l i a n a c y t o s o l i ct R N A ph e, a s d e m o n s t r a t e d b y in vitro a m i n o a c y l a t i o n a n d invivo t r a n s i e n t e x p r e s s i o n t e s t s [ 7 7 ] . U n f o r t u n a t e l y , f a i l u r et o d e t e c t e x p r e s si o n o f t h e c o r r e s p o n d i n g g e n e c o n s t r uc t s i nt r a n s g e n i c t o b a c c o p l a n t s h a s p r e v e n t e d u s f r o m e s t a b -l i s h in g w h e t h e r i t s n e w a l a n i n e i d e n t i t y e n a b l e s t h i s m o d i -f i e d t R N A p he t o b e i m p o r t e d i n t o m i t o c h o n d r i a .

Expression of a chimeric mitochondrial precursor form ofyeost cytosolic aspartyl-tRNA synthetase in transge nic plantsA s a g e n e r a l r u l e , m i t o c h o n d r i a l a m i n o a c y l - t R N A s y n t h e -t a s es a r e a l l n u c l e a r l y e n c o d e d , b u t o n l y t h e i m p o r t e d c y t o -s o l i c t R N A s c a n b e a m i n o a c y l a t e d b y t h e c o r r e s p o n d i n gm i t o c h o n d r i a l e n z y m e s ; n o n - i m p o r t e d c y t o s o l i c t R N A s a r en o t r e c o g n i z e d e f f i c i e n t l y b y t h e m i t o c h o n d r i a l e n z y m e s .W e d e c i d e d t o t r y a n d s e t u p a s i t u a t io n i n w h i c h t r a n s g e n i cp l a n t s w e r e p r o v i d e d w i t h a n a d d i t i o n a l a m i n o a c y l - t R N As y n t h e ta s e a b l e b o t h t o b e i m p o r t e d i n t o m i t o c h o n d r i a a n dt o r e c o g n i z e a n o r m a l l y n o n - i m p o r t e d c y t o s o l i c t R N As p e c i e s . T h e p u r p o s e w a s t o d e t e r m i n e w h e t h e r t h i s w a ss u f f i c ie n t t o d r i v e a n e w t R N A i n t o m i t o c h o n d r i a . W e c h o s ey e a s t a s p a r t y l - t R N A s y n t h e t a s e ( A s p R S ) a n d tR N A A s p f o rt h e s e e x p e r im e n t s , b e c a u s e i n m i t o c h o n d r i a o f b o th m o n o c -o t y l e d o n o u s a n d d i c o t y l e d o n o u s p l a n t s , t R N A A s p i s a n a t i v e ,m i t o c h o n d r i a l l y e n c o d e d s p e c i e s [ 1 8 , 8 0 ] a n d c y t o s o l i c p o -t a t o t R N A A so i s a g o o d s u b s t r a te f o r p u r i f i e d y e a s t c y t o s o l i cA s p R S ( f i g 2 ) .

A t r a n s l a t i o n a l f u s i o n b e t w e e n t h e f i r s t 9 0 a m i n o a c i d s( i n c l u d i n g t h e m i t o c h o n d r i a l t a r g e t i n g t r a n s it p e p t i d e ) o f t h e13- subunit of the Nic o t ia n a p lu m b a g in i f o l i a m i t o c h o n d r i a lA T P s y n t h a s e [ 2 5 ] a n d y e a s t c y t o s o l i c A s p R S [ 2 4 ] w a s e x -p r e s s e d i n t r a n s g e n i c p o t a t o p l a n t s . M i t o c h o n d r i a l p r o t e i na n d t R N A f r a c t i o n s w e r e p r e p a r e d f r o m b o t h t u b e r s a n dl e a v e s o f t h e t r a n s g e n i c l i n e s . P r o t e i n f r a c t i o n s w e r e t e s t e df o r th e p r e s e n c e o f t h e y e a s t A s p R S b y W e s t e rn b l o t ti n g . As i g n a l c o r r e s p o n d i n g t o a p r o t e i n o f t h e e x p e c t e d s i z e ( f i g3 ) w a s s p e c i f i c a l l y o b t a i n e d w i t h m i t o c h o n d r i a l e x t r a c t sf r o m p l a n t s c a r r y i n g t h e y e a s t t r a n s g e n e , a s c o m p a r e d t oc o n t r o l p l a n t s . T h i s b a n d w a s t a k e n t o b e p r o o f o f b o t he x p r e s s io n a n d m i t o c h o n d r i a l im p o r t o f t h e y e a s t A s p R S i nt r a n s g e n i c p o t a t o . T r a n s f e r R N A f r a c t i o n s p r e p a r e d f r o mt h e s a m e m i t o c h o n d r i a l s a m p l e s w e r e t h e n t e s te d b y N o r t h -e r n b l o~ t in g f o r t h e p r e s e n c e o f t h e c y t o s o l i c t R N A A s p u s i n ga l a b e l e d o l i g o n u c l e o t i d e p r o b e d e r i v e d f r o m t h e s e q u e n c eo f t h e s o y b e a n n u c l e a r g e n e e n c o d i n g t h i s t R N A [8 1 ] . D e s -p i t e t h e r e p r o d u c i b l e p r e s e n c e o f t h e y e a s t A s p R S i n t h em i t o c h o n d r i a l f r a c t io n s , n o s i g n i f i c a n t c o - i m p o r t o f t h e c y -t o s o l i c t R N A A s p w a s e v e r d e t e c t e d .

525

Z 0 ,20

~ 0 , 1 00

o ~

!

0 , 0 0 ~ ~ . . a - - _ _ , . _ _ _0 10 20 30

T i m e ( r a i n )Fig 2 . A minoacylat ion k inetics of to tal potato cytoplasmic tRNA(3 mg/m L) in the presence of a saturat ing amount of pur if ied yeasAspRS (20 ~tg /mL).

mr(kDa) 1 297

67

45AspRS

Fig 3 . In vivo expression and mitochondrial import of yeast cyto-solic As pR S in transgenic po tato p lants . M itochondrial ex tractsfrom control (1) and transgenic (2) potato tubers were fractionatedby polyacrylamide gel electrophoresis and analyzed by Westernblotting with a polycional an tiserum raised against yeast cytosolicAspRS. Samples corresponding to equivalent protein amounts (asdetermined acco rding to [91 ]) were load ed. The migration of pro-tein molecular m ass m arkers is indicated. The position of purifiedyeast cy tosolic A spR S is show n by an ar row. The apparent d if -ference in size betwee n purified yeast AspRS a nd the protein spe-cifically detected by the antiserum against AspR S in mitocho ndrialextracts of ~ransgenic potato is likely to be due to ex tra amino acidresidues located downstream of the processing site. as the miaochondr ial presequence of the N plum baginifolia FbATP synthase[~-subunit corresponds to 55 ou t of the 90 residue s encoded by theupstream plant sequence in the chimer ic transgene [92] and s ixamino acids are encoded by the l inker sequence.


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526The o utcome o f these exper iments implies that recogni-t ion by a mitochondr ial ly impor ted aminoacyl- tRNA syn-the tase i s no t neces sa r i ly enough to p rom ote e f f i c ien timport of a tRNA . Th e yeast mitocho ndrial LysRS precursoralone is not sufficient to drive impo rt of tRNAt-y~ into iso-lated yeast mitochondria [72]. However, i t cannot be ex-

cluded at the prese nt s tage that the ex pression level of theyeast AspRS construc t in our transgen ic potato plants wastoo low to allow a detectable tRNAAsp import or that, incontrast to the no rma l form ( ' ,f yeast cyto solic AspRS, th echimer ic precursor form o f th is enzym e in troduced in to thetransgenic plants does not e~ficiently recognize the planttRNAAsp.

Na t iv e t r n Fu 4Liverworl

] t r n F ]C 4 - . ~ U 4. ~ e r a , polato

expression Larch

Conclus ions and prospectsTh e differe nt orig ins of tRNA 'he, tRN A Hi~ and tRNACy~ invarious plants il lustrate well the strategies develo ped byplant m itochondr ia dur ing evolut ion to maintain a co mpletetRNA set (fig 4). These tRNAs are native mitochondrialspecies needing no editing in the primitive, non-vascularplant liverwort, a bryophyte. In larch (gy mn osperm ) mito-chondria, the n ative tRN A His gen e is still e xpre ssed but s e-quen ce divergenc e mak es it necessary to edit the tRNA. Inboth dicotyledono us and some mo nocotyledon ous angios-perms, th is nat ive g en e has b een lo st an d tR NA HL, is ob-t a i n e d f r o m a p l a s t i d - d e r i v e d g e n e a c q u i r e d b y t h emitochondrial genome. Finally, in the monocotyledonousangiosperm wheat , the m itochondr ial genom e does not codefor a tRNA ais and wheat m itochondr ia impor t th is tRNAfrom the cytosoi. Similarly, following sequenc e divergen ce,the native tRNAPh is maintaine d in m itochon dria of dico-tyledonous plants thanks to editing, but has been replacedby a plastid-de rived species in mo noco tyledo nou s plantsand is imported from the cytosol in larch 1341. In the sam ew a y , d e p e n d i n g o n t h e p l a n t l i n e a g e , m i t o c h o n d r i a ltRNACys correspo nds to e ither an ed ited native, a chloro-plast-like or perhaps a nuclearly encoded species (fig 4).Interestingly. altho ugh liverwort rem ains the root species inall cases, the trees based on the gen etic origin and editingof mitoch ondrial tRNAPhe, tRNAHi~ and tRNACy~ do notcorrespond to the phyiogenet ic t ree of the d if ferent p lantspecies conside red. The phylo gene tic tree wo uld first sep-a ra te gym nosperm s ( l a r ch ) f rom ang iosperm s and sub-sequen t ly sp l i t ang iosperm s in to d ico ty ledo nous p lan t s(bean , oeno thera , po ta to , sun f lower ) and m onoco ty le -donous plants (maize, wheat).

I t is evident from the available data that the mitochon-drial tRNA species falling into each of the different ca-tegories (native, e dited native, plastid-derived and importedfrom the cytosol) d if fer to some extent w ith in and betweenplant lineages, However, there is a clear evolutionary tend-ency towards loss of nat ive tRNA genes , which drop innum ber from 2 9 in liverwort [8] to 12 in petunia (Pe t tmtahybr ida , ano ther d icotyled onous plant) [111, 11 in sun-

C 6, e l 2 . C 4 ~ U 6, ILl12,U411Larch

Na t iv e t r n H - J r - " 1U6, UI2, U41 ~ / .,1,] Cp-l ike t rnH [iv 2 ,,/ e r w or l ~ [ " - . .~ . . . . , , , . ,z , .~ , .s " Pota io , unflower,maize

Loss o[ nat ive geneexpression Wheal

Native trnCU28Liverworl

I t rnC ]I C 2 8 ~ U 2 ~

Maize,wheat, ~ . 3~"~NucleartrnC?"Loss of hativegene !expression Larch

Fig 4. Evolution of the gene~ ( trnF, t rnHand m a C ) coding formitochondrial RNAme, tRNA tls and tRNA~-ysas an illustrationofthe strategiesdevelopedby plant mitochondria o keep a com pleteset of functional RNA s. Existenceof editing even ts, expressionofchloroplast-like RN A genes and import of nu cleus-encoded RNAspecies are num bered !, 2 and 3 respectively.Cp-like stands forchloroplast-like. All relevant references are cited in the text or inthe legends o table I and figure I,

f lower [12] and 10 in maize Ii3 ] for instance. I t is interes-t ing to con s ider the o rder of events invo lved in the replace-m e n t o f n a t i v e t R N A s . A s m i t o c h o n d r i a l g e n o m e s a r e


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pr e se nt in m ul t ip le c op ie s pe r c e l l , inac t iva t ion or lo ss o f at R N A g e n e f r o m a p ro p o r ti o n o f th e g e n o m e s i s n ot i m m e -d ia tely l e tha l to the c e l l , but th i s may subse q ue nt ly c r e a te ase lec t ion p re ssure towards a l te rna t ive mod es o f express ion .Conve r se ly , the mi tochondr ia may f i r s t a cqu i re the a l~ema-f ive tRNA (e i the r impor ted o r ch lorop la s t - l ike ) and on lys u b s e q u e n t l y l o s e e x p r e ss i o n o f t h e n a t i v e t RN A . T h i s l a t t e rs e q u e n c e o f e v e n t s m o r e c l o s e l y r e s e m b l e s t h a t i n fe r re d f o rthe tr ansfe r o f pr o te in -c od ing ge n e s to the nuc le us [82 ] .T he fac t tha t r e p lac e me nt o f na t ive tR N A s by nuc le ar lye nc od e d tR N A s d i f fe r s in d if fe re nt p lant l ine age s r a i se s thep r o b le m o f h o w t h e s e l ec t i v it y o f t h e i n t p o r t p r o c e s s c ; nvar y in th i s w ay . I f am inoac y l - tR N A synthe ta se s ar e the k , 'yc ompone nts for impor t spe c i f i c i ty , the n the mi toc hondr ia lp r e c u r s o r s o f t h e s e e n z y m e s w o u l d b e e x p e c t e d t o d i f f e rb e t w e e n p l a n t s w h i c h i m p o r t a g i v e n c y t o s o f i c t R N A i n t omi toc hondr la and those w hic h do no t . E xte ns ive s tud ie s onp l a n t a m i n o a c y l - t R N A s y n t h e t a s e s , s t i l l a p o o r l y d o -c ume nte d domain , w i l l be o f gr e a t he lp in answ e r ing the seq u e s t i o n s . C l o n e d a m i n o a c y l - t R N A s y n t h e t a s e g e n e sshould a l so pr ov ide too l s for pr oduc ing mi toc hondr ia l pr e -c ur sor for ms o f the se e nz ym e s in lar ge quant i ti e s . Suc h pr e -c ur sor s might be r e quir e d for the r c c onst i tu t ion o f tR N Aimpor t w i th i so la te d mi toc hondr ia .D e c ip he r ing the p lant mi toc hondr ia l tR N A impor t m e c h-a n is m m a y , i n t h e lo n g t e r m , y i e ld k n o w l e d g e a n d m o l e c u l a rtoo l s o f pr ac t ic a l be ne f i t. T h e poss ib i l i ty o f tar ge t ing R N A str ansc ribe d fr om n uc le ar transge ne s to mi toc hondr ia w ou ldbe a w ay o f t r ansfe r ring nove l ge ne t i c in format ion in to the seor gane l l e s . Ge ne t i c t r ansfor mat ion o f p lant mi toc hondr iahas ye t to be ac h ie ve d . D e f in ing the ke y c ompone nts o fmi toc hondr ia l tR N A imp or t in p lants and o the r or gan i smsmay a l so lay the foundat ions for the ge ne t i c t r e a tme nt o fpa t i e nts su f fe r ing fr om c y topath ie s due to po int muta t ionsin mi toc hondr ia l tR N A ge n e s , the r a t iona le be ing nuc le are xpr e ss ion , tar ge ting and mi toc hondr ia l imp or t o f a func -t iona l tR N A . A l though i t i s ge ne r a l ly c ons ide r e d tha t nu-c l e ar ly e nc ode d tR N A spe c ie s ar e no t na tur a l ly impor te dinto mam mal ian , and e spe c ia l ly hu man, m i toc hondr ia , the r ei s s o m e e v i d e n c e f o r t h e i m p o r t o f t h e R N A m o i e t y o f t h em i t o c h o n d r i a l R N A p r o c e s s in g e n d o n u c l e a s e ( M R PR N a s e ) (e g [ 8 3 ] ) a n d o f t h e H I V R N A [ 8 4 ] , s u g g e s t i n g t h a tthe pa thw ay pote nt ia l ly e x i s t s and might be e xp lo i te d bysupple me nt ing human c e l l s w i th the appr opr ia te he te r o lo -gnu s c om pon e nts . In addi t ion , r e ce nt e xpe rime nta l e v id e nc eimpl i e s impor t o f a c y toso l i c tR N A into the mi toc hondr iao f mar supia l s (M 0r l M , P~ iabo S , pe r sona l c om m unic a t ion) .

A c k n o w l e d g m e n t sT h e a u t h o r s a r e v e r y g r a t e f u l t o J G a n g l o f f ~m d G E r i an i l b r t h e g i f to f t h e y e a s t AspRS g e n e , p u r e y e a s t A s p R S a n d a n a n t i s e r u ma g a i n s t y e a s t A s p R S a n d t o L W i U m i t z e r f o r th e g i f t o f t h e p a t a t inp r o m o t er . W e w i s h t o t h an k o u r c o - w o r k e r s w h o a r e o r h av e b e e ni n v o l v e d i n th e s e s t u d i e s o n p l a n t m i t o c h o n d r i a l t R N A s : L C h a v a n t ,

527T D e s p r e z , A M D u c h ~ n e - L o u a r n , G G r e e n , S Kauflmann, D R a m a -m o n j i s o a , C R e m a c l e , G S o u c i e t , H W m t z ( S t r a s b o u r g ) a n d KA k a m a , K A k a s h i , V T C C a r n e i r o , N C h o i s n e , R K u m a r , DLance l in , H M ireau (Versa il les ). Tha nks a re a l so due to P Kel tz , PKle th i and R W agn er ( IBMP, S t rasbou rg) fo r ca re o f p lan ts in theg r e e n h o u s e . T h i s w o r k w a s f u n d e d b y t h e t n s t i t u t N a t i o n a l ' d e l aR e c h e r c h e A g r o n o m i q u e ( 1 N R A ) , t h e C e n l r e N a t i o n a l d e l a R e -c h e r c h e S c i e n t i f i q u e ( C N R S ) a n d t h e U n i v e r s i t 6 L o u i s P a s t e u r( U L P , S t r a s b o u r g ) .

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